Polypeptides or proteins may be made by recombinant DNA technology using bacterial cells (e.g. Escherichia coli) as hosts. Thus, bacterial cells may be transformed with plasmid DNA encoding said polypeptide or protein. The bacteria are thereby enabled to express quantities of the polypeptide in either the cytoplasm, or the periplasm or the extracellular space. As the bacteria can be grown in large amounts using large-scale fermentation processes, it is possible to produce large quantities of the polypeptide in this way.
Secretion of polypeptides or proteins into the periplasm has a number of potential advantages, including separation from cytoplasmic proteins, particularly proteases, avoidance of cytoplasmic toxicity, avoidance of N-terminal methionine extension, and accumulation in a more oxidizing environment where disulfide-bond formation may proceed and the protein may fold into a soluble, biologically active confirmation. Superficially, it requires only that the desired protein be fused to a signal peptide (secretory leader) at its N-Terminus although, the efficiency of secretion is likely also influenced by structural features of the protein, as well as its usual location within the cell.
E. coli is well-equipped to secrete proteins through the cytoplasmic membrane into the periplasm, and this approach has been used widely to direct heterologous proteins out of the cell cytoplasm. It has been particularly successful in enabling the production of biologically active antibody fragments and this approach now challenges the production of whole antibodies in animal cell culture.
Secretion of heterologous proteins is rarely 100% efficient and, in several cases, unprocessed precursor protein with the secretory leader attached accumulates within the cell. This observation suggests that one or more components of the secretory apparatus can limit the export of these proteins.
One notable feature of the literature on protein secretion in E. coli is the frequency of reports in which the protein is recovered directly from the growth medium. This can be highly advantageous, but raises unresolved questions about the underlying mechanisms. There are reports showing that proteins are often released into the medium by non specific leakage from, or lysis of, the cell, and not by specific translocation through the outer membrane. This phenomenon is not understood and is highly protein-sequence specific, being extensive with some antibody fragments and insignificant with other polypeptides.
Secretion of proteins into the periplasm is a useful route and can lead to the rapid isolation of a protein for biological evaluation. Its application on industrial scale is currently limited by the general unavailability of efficient, scaleable methods for selective release of periplasmic proteins from the cell.
If the recovery of a recombinant protein from the periplasm can be achieved without contamination by cytoplasmic proteins, subsequent purification steps are simplified very much, since, for instance in E. coli, only 7 out of the 25 known cellular proteases and about 4-8% of the total cell protein are located in the periplasm (Swamy et al., Baneyx et al.).
There are various frequently used methods for selective release of periplasmic proteins. One is cell permeabilization involving chemicals such as chloroform, guanidine-HCl, Cetyl-trimethyl-ammonium-bromide/CTAB or detergents such as Triton X-100 and glycine. Others are permealization using lysozyme/EDTA treatment or application of osmotic shock. These release methods are suitable also for large scale preparation and have been used in many different modifications on a wide range of expression systems with varying degrees of success.
The state of the art methodology on periplasmic release of proteins is documented for example in the following literature:    Swamy et al., J. Bacteriol. 147, 1027-1033, 1982    Baneyx et al., J. Bacteriol. 173, 2696-2703, 1991    Blight et al., TibTech 12, 450-455, 1994    Barbero et al., Journal of Biotechnology 4, 255-267, 1986    Pierce et al., Journal of Biotechnology 58, 1-11, 1997    French et al., Enzyme & Microbial Technology 19, 332-338, 1996    Naglak et al., Enzyme & Microbial Technology 12, 603-611, 1990    Nossal et al., J. Biol. Chem 241 (13), 3055-3062, 1966    Neu et al., J. Biol. Chem 240, 3685-3692, 1965    Hsiung et al., Bib/Technology 4, 991-995, 1986    Carter et al., Bio/Technology 10, 163-167, 1992    Georgiou et al., Biotechnol. Bioeng. 32, 741-748, 1988    Aristidou et al., Biotechnolgy Letters 15 (4), 331-336, 1993    Chaib et al., Biotechnology Techniques 9 (3), 179-184, 1195    Ames et al., J. Bacteriol. 160, 1181-1183, 1984    Gellerfors et al., J. Pharm. Biomed. Anal. 7, 2, 173-83, 1989    Chapman et al, Nature Biotechnology 17, 780-783, 1999    Voss et al., Biochem. J. 298, 719-725, 1994    WO 01/94585    U.S. Pat. No. 4,845,032    U.S. Pat. No. 4,315,852
Whereas recombinant techniques can be employed to produce high yields of a crude polypeptide, the isolation and purification of the polypeptide requires sophisticated and extensive procedures.
In a typical isolation procedure, the fermentation harvest broth is adjusted to a neutral pH (e.g pH 6.5-7.5) by addition of acid or caustic. Thereafter, the bacterial cells are removed e.g. by centrifugation or microfiltration to leave a liquid supernatant, containing unwanted soluble by-products, which is discarded. The resultant bacterial cell mass is resuspended in an appropriate medium, e.g. a suitable buffer, and the cells are disrupted to extract and isolate the product.
Laborious extraction and isolation procedures are usually carried out in order to separate the polypeptide of interest from as much fermentation by-products and other contaminants as possible to ensure that subsequent purification steps proceed in an as efficient manner as possible.
Purification steps known in the art generally comprise precipitation and chromatographic separation techniques, and sometimes require additional steps like diafiltration and/or concentration procedures, which are laborious and may lead to lower yields of extracted polypeptide and higher production costs.
The extraction, isolation and purification of a polypeptide implicates losses of material or biological activity at every stage of the process.
Proteolysis, i.e. degradation of the polypeptides by proteolytic enzymes, usually occurring after disruption of the bacterial cells, but also observed in vivo, is considered to be one of the main causes of protein loss. This adventitious proteolysis is a technical problem which requires modifications of the methodology to minimize the degradation of the polypeptide of interest.
One way of keeping the rate of proteolysis low, is generally to perform the harvest, extraction, isolation and purification procedures at reasonably low temperatures and as fast as possible. Accordingly, relevant text books and standard protocols on isolation and purification of polypeptides in general teach to proceed without unnecessary delays and interruptions (Protein Protocols, Ed. J. M. Walker, Humana Press Inc., January 1998).
Accordingly, there is a need for novel processes that enable the extraction of recombinant polypeptides of interest from bacterial cells in a high yielding and cost-effective manner.
The present invention complies with the above mentioned needs by providing novel methods for the preparation of recombinant polypeptides.
In the context of the present invention it has surprisingly been found that the yield of a recombinant polypeptide expressed in a bacterial host comprising a periplasm can be increased by interrupting the isolation process after fermentation and put on hold the further processing of the fermentation harvest broth before the subsequent steps of extraction and isolation of said polypeptide are performed.